Phosphorylation occurs in majority of signal transduction cascades, and plays pivotal roles in cellular functions such as proliferation, differentiation, metabolism and apoptosis. Kinases, which catalyze phosphorylation of proteins, peptides, lipid and other substrates, have been the focus for both pharmaceutical industry and research laboratories. Since the discovery that many oncogenes encode protein kinases in mid 70s, great efforts have been made to develop kinase regulators that may be useful for treating diseases associated with aberrant kinase functions. The efforts have led to successful development of a number of small molecule kinase regulators. Gleevec, a small molecule inhibitor specific for tyrosine kinase c-abl, was approved by FDA for use in treating chronic myeogeneous leukemia. Irresa and Tarceva, both small molecule inhibitors of EGFR kinase, were approved by FDA in 2003 and 2005, respectively, for use in treating non-small cell lung carcinoma. Bay43, a small molecule inhibitor of raf kinase, was approved by FDA for use in treating kidney cancer. The success has fueled more efforts for the drug discovery industry to search for more kinase inhibitors in order to treat more diseases. These efforts all start with development of appropriate kinase assays amenable for high throughput screening.
The human genome encodes for 518 protein kinases that are responsible for the phosphorylation of 30% of cellular proteins. In addition to protein kinases, lipid kinases and sugar kinases play equally important roles in cellular functions. On one hand, each kinase has a unique substrate, be it a peptide, protein, lipid or carbohydrate. On the other hand, all kinases use ATP and generate ADP.
Besides kinases, there are many other ADP-generating enzymes are or clinical importance. Particularly, an ATPase activity is associated with many different types of proteins, including molecular chaperones, myosin, kinesins, and transporter proteins, many of them are emerging drug targets. Disruption of microtubule function and/or assembly is one approach that is being pursued for development of anticancer agents. A good example of molecular chaperone is HSP90. Being required for proper folding and stability of a number of oncogenic “client” proteins (including c-Raf-1, cdk4, ErbB2, and c-Met), HSP 90 is widely pursued as a target for cancer drug discovery.
Most kinase assays are substrate-specific and are developed to measure the incorporation of phosphate into the unique substrates. The classic kinase assays measure the incorporation of gamma-P33 into the substrate. These assays use radioactive isotope, require filtration separation and are low throughput. More recent assays follow the same principle but remove the need for either radioactive isotopes or filtration separation. For example, in some assays, peptide substrates are conjugated with biotin and captured with streptavidin-coated plates. Phosphorylated peptide products are then detected with specific antibodies with colormetric, fluorescent or time-delayed fluorescent readout. In these ELISA type of assays, besides the drawback that washing steps limit throughput, the assays are applied to tyrosine kinases only since, so far, only anti-phosphorylated tyrosine antibodies are available. In SPA assays, streptavidin-coated microbeads impregnated with scintillant are used to capture the biotinylated peptide and measure the incorporation of gamma-P33. An improvement over classic kinase assays is that no filtration separation is needed any more; however, these SPA assays still require the use of radioactive materials. ALPHA screen removes the needs for radioactive materials but requires two beads systems and special instruments. In addition to the cost, its sensitivity to light and temperature variations limit its wide implementation. Microfluidic technologies make it possible to separate and measure phosphorylated products on microchips. However, the requirement for special instrument and the relatively low throughput, again, limits its wide implementation.
Other assays measure organic phosphate to assess kinase activity. Some assays utilize chemicals (such as malachite) that change color upon binding of organic phosphate (Innova, Anaspec and Bioassay system). Other assays utilize enzyme coupling reactions to convert phosphate to chromogenic or fluorogenic signals (Invitrogen). However, due to the presence of phosphate in most biological samples, it is difficult to control background and to achieve desirable sensitivity.
The Kinase-Glo™ assay, developed by Promega, is the first universal kinase assay that targets ATP, the substrate shared by all kinases. The assay is universal because it applies to all kinases and other enzymes that use ATP as a substrate. However, because this assay measures the reduction of ATP, the sensitivity of the assay is limited. The ADP-Quest™ assay, from the company DiscoverX, measures the increase of ADP, the product generated by all kinases. However, the incompatibility of the ADP-Quest™ assay with several reagents commonly used in kinase assays, limits its application. In addition, like all homogeneous assays with fluorescent readout, the ADP-Quest™ assay is vulnerable to interference stemming from fluorescent compounds. In comparison with the Kinase-Glo™ assay, this assay has an improved sensitivity.
With the development of monoclonal antibodies that can distinguish ADP from ATP (affinity for ADP is 100 fold stronger than ATP), Bellbrook made a Transcreener™ kinase assay kit available to the industry. In this assay, ADP generated from a kinase reaction competes with labeled tracers for the antibody. Binding of ADP to its antibody results in dissociation of a tracer, and thus reduction of fluorescent signals. One major drawback of this assay is limitation of sensitivity because a signal reduction is being measured. Another drawback of the assay is that it cannot scale up to a 1536 well format.
Therefore, there is a need for improved kinase assays.